Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. Recently, an antibiotic-resistant strain of this serovar was implicated in a large 2011 multistate outbreak resulting from consumption of contaminated ground turkey that involved 136 confirmed cases, with one death. In this study, we assessed the evolutionary diversity of 44 S. Heidelberg isolates using whole-genome sequencing (WGS) generated by the 454 GS FLX (Roche) platform. The isolates, including 30 with nearly indistinguishable (one band difference) Xbal pulsed-field gel electrophoresis patterns (JF6X01.0032, JF6X01.0058), were collected from various sources between 1982 and 2011 and included nine isolates associated with the 2011 outbreak. Additionally, we determined the complete sequence for the chromosome and three plasmids from a clinical isolate associated with the 2011 outbreak using the Pacific Biosciences (PacBio) system. Using single-nucleotide polymorphism (SNP) analyses, we were able to distinguish highly clonal isolates, including strains isolated at different times in the same year. The isolates from the recent 2011 outbreak clustered together with a mean SNP variation of only 17 SNPs. The S. Heidelberg isolates carried a variety of phages, such as prophage P22, P4, lambda-like prophage Gifsy-2, and the P2-like phage which carries the sopE1 gene, virulence genes including 62 pathogenicity, and 13 fimbrial markers and resistance plasmids of the incompatibility (Inc)I1, IncA/C, and IncHI2 groups. Twenty-one strains contained an IncX plasmid carrying a type IV secretion system. On the basis of the recent and historical isolates used in this study, our results demonstrated that, in addition to providing detailed genetic information for the isolates, WGS can identify SNP targets that can be utilized for differentiating highly clonal S. Heidelberg isolates.
outbreak; antimicrobial resistance; plasmid; SNP analysis; trace-back
A study of prevalence, diversity, and antimicrobial resistance of Salmonella enterica in surface water in the southeastern United States was conducted. A new scheme was developed for recovery of Salmonella from irrigation pond water and compared with the FDA's Bacteriological Analytical Manual (8th ed., 2014) (BAM) method. Fifty-one isolates were recovered from 10 irrigation ponds in produce farms over a 2-year period; nine Salmonella serovars were identified by pulsed-field gel electrophoresis analysis, and the major serovar was Salmonella enterica serovar Newport (S. Newport, n = 29), followed by S. enterica serovar Enteritidis (n = 6), S. enterica serovar Muenchen (n = 4), S. enterica serovar Javiana (n = 3), S. enterica serovar Thompson (n = 2), and other serovars. It is noteworthy that the PulseNet patterns of some of the isolates were identical to those of the strains that were associated with the S. Thompson outbreaks in 2010, 2012, and 2013, S. Enteritidis outbreaks in 2011 and 2013, and an S. Javiana outbreak in 2012. Antimicrobial susceptibility testing confirmed 16 S. Newport isolates of the multidrug resistant-AmpC (MDR-AmpC) phenotype, which exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT), and to the 1st, 2nd, and 3rd generations of cephalosporins (cephalothin, amoxicillin-clavulanic acid, and ceftriaxone). Moreover, the S. Newport MDR-AmpC isolates had a PFGE pattern indistinguishable from the patterns of the isolates from clinical settings. These findings suggest that the irrigation water may be a potential source of contamination of Salmonella in fresh produce. The new Salmonella isolation scheme significantly increased recovery efficiency from 21.2 (36/170) to 29.4% (50/170) (P = 0.0002) and streamlined the turnaround time from 5 to 9 days with the BAM method to 4 days and thus may facilitate microbiological analysis of environmental water.
Multidrug-resistant (MDR) Salmonella enterica subsp. enterica serotype Newport has been a long-standing public health concern in the United States. We present the complete sequences of six IncA/C plasmids from animal-derived MDR S. Newport ranging from 80.1 to 158.5 kb. They shared a genetic backbone with S. Newport IncA/C plasmids pSN254 and pAM04528.
Shiga toxin-producing Escherichia coli (STEC) O26 is the second leading E. coli serogroup responsible for human illness outbreaks behind E. coli O157:H7. Recent outbreaks have been linked to emerging pathogenic O26:H11 strains harboring stx2 only. Cattle have been recognized as an important reservoir of O26 strains harboring stx1; however the reservoir of these emerging stx2 strains is unknown. The objective of this study was to identify nucleotide polymorphisms in human and cattle-derived strains in order to compare differences in polymorphism derived genotypes and virulence gene profiles between the two host species. Whole genome sequencing was performed on 182 epidemiologically unrelated O26 strains, including 109 human-derived strains and 73 non-human-derived strains. A panel of 289 O26 strains (241 STEC and 48 non-STEC) was subsequently genotyped using a set of 283 polymorphisms identified by whole genome sequencing, resulting in 64 unique genotypes. Phylogenetic analyses identified seven clusters within the O26 strains. The seven clusters did not distinguish between isolates originating from humans or cattle; however, clusters did correspond with particular virulence gene profiles. Human and non-human-derived strains harboring stx1 clustered separately from strains harboring stx2, strains harboring eae, and non-STEC strains. Strains harboring stx2 were more closely related to non-STEC strains and strains harboring eae than to strains harboring stx1. The finding of human and cattle-derived strains with the same polymorphism derived genotypes and similar virulence gene profiles, provides evidence that similar strains are found in cattle and humans and transmission between the two species may occur.
Escherichia coli; O26; Shiga toxins; polymorphisms; phylogenetic
Phage typing has been used for the epidemiological surveillance of Salmonella enterica serovar Enteritidis for over 2 decades. However, knowledge of the genetic and evolutionary relationships between phage types is very limited, making differences difficult to interpret. Here, single nucleotide polymorphisms (SNPs) identified from whole-genome comparisons were used to determine the relationships between some S. Enteritidis phage types (PTs) commonly associated with food-borne outbreaks in the United States. Emphasis was placed on the predominant phage types PT8, PT13a, and PT13 in North America. With >89,400 bp surveyed across 98 S. Enteritidis isolates representing 14 distinct phage types, 55 informative SNPs were discovered within 23 chromosomally anchored loci. To maximize the discriminatory and evolutionary partitioning of these highly homogeneous strains, sequences comprising informative SNPs were concatenated into a single combined data matrix and subjected to phylogenetic analysis. The resultant phylogeny allocated most S. Enteritidis isolates into two distinct clades (clades I and II) and four subclades. Synapomorphic (shared and derived) sets of SNPs capable of distinguishing individual clades/subclades were identified. However, individual phage types appeared to be evolutionarily disjunct when mapped to this phylogeny, suggesting that phage typing may not be valid for making phylogenetic inferences. Furthermore, the set of SNPs identified here represents useful genetic markers for strain differentiation of more clonal S. Enteritidis strains and provides core genotypic markers for future development of a SNP typing scheme with S. Enteritidis.
Shiga toxin-producing Escherichia coli (STEC) strains (n = 194) representing 43 serotypes and E. coli K-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains (n = 81) was further analyzed for subtype I-E cas and virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-E cas and virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity (P < 0.05). Fewer spacers were found in strains that had a greater probability of causing outbreaks and disease than in those with lower virulence potential (P < 0.05). The relationship between the CRISPR-cas system and potential virulence needs to be determined on a broader scale, and the biological link will need to be established.
Next-generation sequencing is being evaluated for use with food-borne illness investigations, especially when the outbreak strains produce patterns that cannot be discriminated from non-outbreak strains using conventional procedures. Here we report complete genome assemblies of two Salmonella enterica serovar Heidelberg strains with a common pulsed-field gel electrophoresis pattern isolated during an outbreak investigation.
Aminoglycoside resistance in Campylobacter has been routinely monitored in the United States in clinical isolates since 1996 and in retail meats since 2002. Gentamicin resistance first appeared in a single human isolate of Campylobacter coli in 2000 and in a single chicken meat isolate in 2007, after which it increased rapidly to account for 11.3% of human isolates and 12.5% of retail isolates in 2010. Pulsed-field gel electrophoresis analysis indicated that gentamicin-resistant C. coli isolates from retail meat were clonal. We sequenced the genomes of two strains of this clone using a next-generation sequencing technique in order to investigate the genetic basis for the resistance. The gaps of one strain were closed using optical mapping and Sanger sequencing, and this is the first completed genome of C. coli. The two genomes are highly similar to each other. A self-transmissible plasmid carrying multiple antibiotic resistance genes was revealed within both genomes, carrying genes encoding resistance to gentamicin, kanamycin, streptomycin, streptothricin, and tetracycline. Bioinformatics analysis and experimental results showed that gentamicin resistance was due to a phosphotransferase gene, aph(2″)-Ig, not described previously. The phylogenetic relationship of this newly emerged clone to other Campylobacter spp. was determined by whole-genome single nucleotide polymorphisms (SNPs), which showed that it clustered with the other poultry isolates and was separated from isolates from livestock.
Over 90% of the human listeriosis cases are caused by Listeria monocytogenes serotypes 1/2a, 1/2b and 4b strains. As an alternative to antigen-antibody based serotyping, a PCR-based method for serogrouping has been developed and validated. In this communication, we report an in-depth analysis of five 4b variant strains, four clinical isolates from Australia and one environmental isolate from USA. Although these five strains were serotype 4b by classical serotyping method, the serogrouping PCR profiles of these strains show the presence of a 1/2a-3a specific amplicon in addition to the standard 4b-4d-4e specific amplicons. These strains were further analyzed by pulsed field gel electrophoresis, binary gene typing, multi-locus variable-number-tandem-repeat analysis and a high density pan-genomic Listeria microarray. Using these sub-typing results, the clinical isolates were grouped into two distinct genomic groups- one of which could be part of an unidentified outbreak. The microarray results when compared with our database of other 4b outbreak isolates indicated that the serotype 4b variant strains represent very different genotypic profiles than the known reported 4b outbreak strains representing major epidemic clones. The acquisition of serotype 1/2a gene clusters by the 4b variant strains appears to be independent in origin, spanning large areas of geographical and temporal space and may indicate predisposition of some 4b strains towards accepting DNA from related organisms.
Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of salmonellosis. Here, we report a closed genome sequence, including sequences of 3 plasmids, of Salmonella serovar Typhimurium var. 5− CFSAN001921 (National Antimicrobial Resistance Monitoring System [NARMS] strain ID N30688), which was isolated from chicken breast meat and shows resistance to 10 different antimicrobials. Whole-genome and plasmid sequence analyses of this isolate will help enhance our understanding of this pathogenic multidrug-resistant serovar.
Pathogenicity islands (PAIs) play an important role in Shiga toxin-producing Escherichia coli (STEC) pathogenicity. The distribution of PAIs OI-122, OI-43/48, and OI-57 and a high-pathogenicity island (HPI) were determined among 98 STEC strains assigned to seropathotypes (SPTs) A to E. PCR and PCR-restriction fragment length polymorphism assays were used to identify 14 virulence genes that belonged to the four PAIs and to subtype eae and stx genes, respectively. Phylogenetic trees were constructed based on the sequences of pagC among 34 STEC strains and iha among 67 diverse pathogenic E. coli, respectively. Statistical analysis demonstrated that the prevalences of OI-122 (55.82%) and OI-57 (82.35%) were significantly greater in SPTs (i.e., SPTs A, B, and C) that are frequently associated with severe disease than in other SPTs. terC (62.5%) and ureC (62.5%) in OI-43/48 were also significantly more prevalent in SPTs A, B, and C than in SPTs D and E. In addition, OI-122, OI-57, and OI-43/48 and their associated virulence genes (except iha) were found to be primarily associated with eae-positive STEC, whereas HPI occurred independently of the eae presence. The strong association of OI-122, OI-43/48, and OI-57 with eae-positive STEC suggests in part that different pathogenic mechanisms exist between eae-positive and eae-negative STEC strains. Virulence genes in PAIs that are associated with severe diseases can be used as potential markers to aid in identifying highly virulent STEC.
Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.
Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are emerging food-borne pathogens causing life-threatening diseases and food-borne outbreaks. A better understanding of their evolution provides a framework for developing tools to control food safety. We obtained 15 genomes of non-O157 STEC strains, including O26, O111, and O103 strains. Phylogenetic trees revealed a close relationship between O26:H11 and O111:H11 and a scattered distribution of O111. We hypothesize that STEC serotypes with the same H antigens might share common ancestors.
Shiga toxin-producing Escherichia coli (STEC) causes severe illness in humans, including hemorrhagic colitis and hemolytic uremic syndrome. A parallel evolutionary model was proposed in which E. coli strains of distinct phylogenies independently integrate Shiga toxin-encoding genes and evolve into STEC. We report the draft genomes of two emerging non-O157 STEC strains.
Salmonellosis is a major contributor to the global public health burden. Salmonella enterica serotype Newport has ranked among three Salmonella serotypes most commonly associated with food-borne outbreaks in the United States. It was thought to be polyphyletic and composed of independent lineages. Here we report draft genomes of eight strains of S. Newport from diverse hosts and locations.
Salmonella Newport has ranked in the top three Salmonella serotypes associated with foodborne outbreaks from 1995 to 2011 in the United States. In the current study, we selected 26 S. Newport strains isolated from diverse sources and geographic locations and then conducted 454 shotgun pyrosequencing procedures to obtain 16–24 × coverage of high quality draft genomes for each strain. Comparative genomic analysis of 28 S. Newport strains (including 2 reference genomes) and 15 outgroup genomes identified more than 140,000 informative SNPs. A resulting phylogenetic tree consisted of four sublineages and indicated that S. Newport had a clear geographic structure. Strains from Asia were divergent from those from the Americas. Our findings demonstrated that analysis using whole genome sequencing data resulted in a more accurate picture of phylogeny compared to that using single genes or small sets of genes. We selected loci around the mutS gene of S. Newport to differentiate distinct lineages, including those between invH and mutS genes at the 3′ end of Salmonella Pathogenicity Island 1 (SPI-1), ste fimbrial operon, and Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) associated-proteins (cas). These genes in the outgroup genomes held high similarity with either S. Newport Lineage II or III at the same loci. S. Newport Lineages II and III have different evolutionary histories in this region and our data demonstrated genetic flow and homologous recombination events around mutS. The findings suggested that S. Newport Lineages II and III diverged early in the serotype evolution and have evolved largely independently. Moreover, we identified genes that could delineate sublineages within the phylogenetic tree and that could be used as potential biomarkers for trace-back investigations during outbreaks. Thus, whole genome sequencing data enabled us to better understand the genetic background of pathogenicity and evolutionary history of S. Newport and also provided additional markers for epidemiological response.
Salmonella enterica is recognized as one of the most common bacterial agents of foodborne illness. We report draft genomes of four Salmonella serovar Heidelberg isolates associated with the recent multistate outbreak of human Salmonella Heidelberg infections linked to kosher broiled chicken livers in the United States in 2011. Isolates 2011K-1259 and 2011K-1232 were recovered from humans, whereas 2011K-1724 and 2011K-1726 were isolated from chicken liver. Whole genome sequence analysis of these isolates provides a tool for studying the short-term evolution of these epidemic clones and can be used for characterizing potentially new virulence factors.
Salmonella enterica serovar Heidelberg has caused numerous outbreaks in humans. Here, we report draft genomes of five isolates of serovar Heidelberg associated with the recent (2011) multistate outbreak linked to ground turkey in the United States. Isolates 2011K-1110 and 2011K-1132 were recovered from humans, while isolates 2011K-1138, 2011K-1224, and 2011K-1225 were recovered from ground turkey. Whole-genome sequence analysis of these isolates provides a tool for studying the short-term evolution of these epidemic clones.
Escherichia coli O104 isolates collected from different sources in the United States were examined for virulence genes typical of enterohemorrhagic E. coli and those identified in the O104:H4 isolate associated with the 2011 German outbreak. The unexpected presence of virulence markers in these isolates highlights the importance of screening unusual and potentially pathogenic Shiga toxin-producing E. coli serotypes.
Campylobacter jejuni is a major zoonotic pathogen. A highly virulent, tetracycline-resistant C. jejuni clone (clone SA) has recently emerged in ruminant reservoirs and has become the predominant cause of sheep abortion in the United States. To determine whether clone SA is associated with human disease, we compared the clinical isolates of clone SA from sheep abortions with the human isolates of the PulseNet National Campylobacter databases at the CDC and the FDA using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and serotyping. The combined SmaI and KpnI PFGE pattern designations of clone SA from sheep were indistinguishable from those of 123 (9.03%) human C. jejuni isolates (total, 1,361) in the CDC database, among which 56 were associated with sporadic infections and 67 were associated with outbreaks that occurred in multiple states from 2003 to 2010. Most of the outbreaks were attributed to raw milk, while the sources for most of the sporadic cases were unknown. All clone SA isolates examined, including PFGE-matched human isolates, belong to sequence type 8 (ST-8) by MLST and serotype HS:1,8, further indicating the clonality of the related isolates from different host species. Additionally, C. jejuni clone SA was identified in raw milk, cattle feces, the feces and bile of healthy sheep, and abortion cases of cattle and goats, indicating the broad distribution of this pathogenic clone in ruminants. These results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans and indicate that C. jejuni clone SA is an important threat to public health.
Recent studies indicate that chronic ouabain treatment leads to hypertension and hypertensive vascular remodeling. Grape seed proanthocyanidin extract (GSPE) has been reported to be effective in treating arteriosclerosis, while little is known about its effect on systolic blood pressure and vascular remodeling. In this study, the effects of GSPE on systolic blood pressure and vascular remodeling were analyzed by treating ouabain-induced hypertensive rats with GSPE (250 mg/kg·d). The expression of nitric oxide (NO) and endothelin-1 (ET-1) in thoracic aorta was examined by ELISA; the mRNA and protein levels of TGF-β1 were detected using real-time PCR and western blotting, respectively. The results showed that the systolic blood pressure was significantly decreased following treatment with GSPE, with blocked vascular remodeling. The ET-1 content was reduced while NO production was increased in the GSPE group, which showed improved vascular endothelial function. Moreover, GSPE also reduced TGF-β1 expression in the thoracic aorta, which is a determinant in vascular remodeling. In conclusion, GSPE antagonized ouabain-induced hypertension and vascular remodeling and is recommended as a potential anti-hypertensive agent for patients with hypertensive vascular diseases.
grape seed proanthocyanidin extract; ouabain; rats; hypertension; vascular remodeling
Determining drug resistance trends will optimize treatment and public health responses.
We conducted a retrospective study of Escherichia coli isolates recovered from human and food animal samples during 1950–2002 to assess historical changes in antimicrobial drug resistance. A total of 1,729 E. coli isolates (983 from humans, 323 from cattle, 138 from chickens, and 285 from pigs) were tested for susceptibility to 15 antimicrobial drugs. A significant upward trend in resistance was observed for ampicillin (p<0.001), sulfonamide (p<0.001), and tetracycline (p<0.001). Animal strains showed increased resistance to 11/15 antimicrobial agents, including ampicillin (p<0.001), sulfonamide (p<0.01), and gentamicin (p<0.001). Multidrug resistance (≥3 antimicrobial drug classes) in E. coli increased from 7.2% during the 1950s to 63.6% during the 2000s. The most frequent co-resistant phenotype observed was to tetracycline and streptomycin (29.7%), followed by tetracycline and sulfonamide (29.0%). These data describe the evolution of resistance after introduction of new antimicrobial agents into clinical medicine and help explain the range of resistance in modern E. coli isolates.
Escherichia coli; bacteria; antimicrobial drug resistance humans; food animals; United States
Due to emerging resistance to traditional antimicrobial agents, such as ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol, azithromycin is increasingly used for the treatment of invasive Salmonella infections. In the present study, 696 isolates of non-Typhi Salmonella collected from humans, food animals, and retail meats in the United States were investigated for antimicrobial susceptibility to azithromycin. Seventy-two Salmonella enterica serotype Typhi isolates from humans were also tested. For each isolate, MICs of azithromycin and 15 other antimicrobial agents were determined by broth microdilution. Among the non-Typhi Salmonella isolates, azithromycin MICs among human isolates ranged from 1 to 32 μg/ml, whereas the MICs among the animal and retail meat isolates ranged from 2 to 16 μg/ml and 4 to 16 μg/ml, respectively. Among Salmonella serotype Typhi isolates, the azithromycin MICs ranged from 4 to 16 μg/ml. The highest MIC observed in the present study was 32 μg/ml, and it was detected in three human isolates belonging to serotypes Kentucky, Montevideo, and Paratyphi A. Based on our findings, we propose an epidemiological cutoff value (ECOFF) for wild-type Salmonella of ≤16 μg/ml of azithromycin. The susceptibility data provided could be used in combination with clinical outcome data to determine tentative clinical breakpoints for azithromycin and Salmonella enterica.